Description
Citrate Assay Kit (Colorimetric or Fluorometric) (BA0090) (BA0090)
The Citrate Assay Kit (Colorimetric or Fluorometric) (SKU: BA0090) provides a simple, automation-ready procedure for measuring citrate concentration. Citrate is an intermediate in the citric acid cycle and is involved in fatty acid synthesis. In this assay, citrate is converted into pyruvate, which is then oxidised with the conversion of a dye into a coloured and fluorescent form. The colour intensity at 570 nm, or fluorescence intensity at excitation/emission 530/585 nm, is directly proportional to the citrate concentration in the sample. The homogeneous mix-incubate-measure format can be readily automated for high-throughput analysis.
| Product Name: | Citrate Assay Kit (Colorimetric or Fluorometric) (BA0090) |
| SKU: | BA0090 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 4 to 400 uM; fluorometric 0.5 to 40 uM citrate |
| Sample Type: | Plasma, serum, urine, tissue and culture media |
| Species Reactivity: | All |
| Assay Time: | 15 minutes at room temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric determination of citrate. Citrate is converted to pyruvate, which is oxidised to convert a dye to a coloured form read at 570 nm or by fluorescence at excitation/emission 530/585 nm.
- Fast and sensitive; colorimetric range 4 to 400 uM and fluorometric range 0.5 to 40 uM citrate
- Convenient and high-throughput mix-incubate-measure format
- Readily automated on liquid handling systems
- Supports use of an internal standard for challenging matrices
- Citrate determination in biological samples such as plasma, serum, urine, tissue and culture media
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reagent preparation. Dissolve the CL Enzyme with 120 uL Developer, pipetting up and down to ensure it is fully dissolved; reconstituted CL enzyme is stable for 4 weeks at -20C. Resuspend before each use. |
| 2 | Sample preparation. Homogenise tissue or cell samples (2 x 10^6) in 100 uL PBS and centrifuge at 14,000 rpm for 5 minutes; use clear supernatant. Avoid culture media with high pyruvate concentrations. Deproteinate serum and plasma with a 10 kDa spin filter, or measure directly using an internal standard. Dilute urine at least 5-fold and use an internal standard. |
| 3 | Standards (colorimetric). Dilute the citrate standard to 400 uM by mixing 10 uL of 10 mM standard with 240 uL distilled water, then dilute as shown in the dilution table. Transfer 20 uL of each standard to separate wells. |
| 4 | Samples. Add 20 uL of each sample to two separate wells (each sample requires a sample blank). If using an internal standard, prepare a 1000 uM citrate standard and set up three reactions per sample: sample plus standard (5 uL 1000 uM citrate + 20 uL sample), sample alone and sample blank (5 uL distilled water + 20 uL sample). |
| 5 | Citrate detection. Prepare working reagent by mixing, per reaction, 85 uL Developer, 1 uL CL Enzyme, 1 uL ODC Enzyme and 1 uL Dye Reagent; for sample blanks, prepare working reagent without the CL Enzyme. Add 80 uL of the appropriate working reagent to each standard and sample well, mix and incubate protected from light for 15 minutes at room temperature. Read OD570nm. |
| 6 | Fluorometric procedure. Dilute the colorimetric standards 1:10 in distilled water (range 1 to 40 uM). Transfer 20 uL standards and samples into a black 96-well plate, add 80 uL appropriate working reagent, incubate protected from light for 15 minutes and read fluorescence at excitation/emission 530/585 nm. |
Subtract the blank value (#4) from the standard values and plot dOD or dF against standard concentrations to determine the slope. [Citrate] = (R-sample - R-blank) / slope x n (uM). If an internal standard was used, [Citrate] = (R-sample - R-blank) / (R-standard - R-sample) x ([Standard]/4) x n (uM), where the internal standard concentration is divided by 4 because its volume is four times lower than the sample. Conversions: 100 uM citrate equals 19.1 mg/L, 0.0019% or 19.1 ppm. If the calculated concentration exceeds 400 uM (colorimetric) or 40 uM (fluorometric), dilute the sample and repeat.
| Component | Quantity | Storage |
| Developer | 10 mL | -20C |
| Dye Reagent | 120 uL | -20C |
| Citrate Standard | 500 uL | -20C |
| CL Enzyme | Dried | -20C |
| ODC Enzyme | 120 uL | -20C |
